Tuberculosis (TB)
The increase in incidence in bovine tuberculosis, caused by infection with Mycobacterium bovis, in developed countries such as the UK, New Zealand and the USA, poses significant economic problems and increasing risks to human health. Despite continued use of the skin test and slaughter policy in the UK, the incidence of bovine TB in UK cattle herds is now increasing rapidly. There is now clearly a requirement for improved diagnostic tests and/or vaccination of cattle to halt the spread of this disease and thus reduce economic losses and risks to human health.
Currently, the two most widely used diagnostic tests for tuberculosis in cattle are based on the detection of established cell mediated immune responses: (a) The delayed type hypersensitivity (DTH) response—detected by the comparative tuberculin skin test (Monaghan et al (1994) Vet Microbiol 40(1-2):111-24); (b) the in vitro synthesis of IFN gamma by whole blood cultured with mycobacterial antigens (PPD), that can be subsequently detected by the Bovigam™ ELISA (Hope et al (2005a) Clin. Exp. Immunol. 139:48-56). However, it is well documented that both diagnostic techniques are only partially successful in identifying infected cattle from normal uninfected animals with reported sensitivity ranging from 68 to 95% for the skin test (Monaghan et al (1994, as above).
Incorrect diagnosis may cause significant problems, for example, where false positives exist, the movement restrictions that are placed on farms with herd breakdowns have significant negative economic impact on those affected farms. Infected cattle that are ‘missed’ by the skin test (false negatives) are a possible source for cattle to cattle transmission of M. bovis. 
In addition these tests are expensive and time consuming, with a minimum of 2 days from initial sampling to diagnosis by ELISA and 3 days by skin testing. There is thus a need for a new, more rapid, test for TB, preferably one which would allow “same day” diagnosis of infected animals.
To date there are no commercial vaccines available for tuberculosis in cattle. The human vaccine strain BCG produces significant protection, particularly in neonates (Hope et al (2005a) as above), but has been shown to have variably efficacy in cattle, as in humans. More recently, alternative vaccination strategies such as neonatal vaccination (Hope et al (2005a) as above; Marchant et al (199) J. Immunol 163:2249-55; Vekemans et al (2001) 31:1531-5) and heterologous prime-boost vaccination (Vordermeier et al (2004) Immunology 112:461-70; McShane et al (2005) Tuberculosis 85:47-52; McShane and Hill (2005) Microbes Infect 7:962-7; and Ferraz et al (2004) 72:6945-50), have significantly improved the efficacy of BCG such that it is now being considered for additional trials in humans and cattle (Hope and Vordermeier (2005) Expert Review of Vaccines 4:891-902).
BCG vaccinated individuals test positive by both the skin test and the standard IFNγ test. Differential diagnosis of BCG vaccinated from M. bovis infected cattle has been achieved through the use of antigens such as ESAT-6 and CFP-10 (van Pinxteren et al (2000) 7:155-60) present in virulent M. bovis but absent from BCG. Reactivity to ESAT-6 and/or CFP10 was shown to correlate with disease severity and could be used to distinguish protected BCG vaccinates from M. bovis infected animals (Vordermeier et al (2002) 70:3026-32; Buddle et al (1999) Clin Dign Lab Immunol. 6:1-5). However, reactivity to these antigens is not observed in all M. bovis infected animals (Pollock et al (1997) J. Infect Dis 175:1251-4) and is significantly affected by prior sensitisation of cattle to environmental mycobacteria (Hope et al (2005b) Clin Exp Immnnol 141:432-9). Thus, more specific antigens, or more sensitive assay systems are required for accurate diagnosis of bovine TB and distinction of vaccinated individuals.
If BCG vaccination of cattle becomes more widespread, it will no longer be possible to use these standard skin and IFNγ tests routinely because they are unsuitable for vaccinated or part-vaccinated herds. There is thus a need for a simple, inexpensive diagnostic test which can distinguish vaccinated subjects from those which are infected with virulent mycobacteria.
Paratuberculosis (ParaTB)
Paratuberculosis (paraTB) which is also known as Johne's disease, is a chronic progressive enteric disease of ruminants caused by infection with Mycobacterium paratuberculosis. ParaTB is widely distributed internationally in domesticated ruminants such as cattle, sheep, goats, as well as deer, antelope and bison. The clinical disease is characterised by chronic or intermittent diarrhoea, emaciation and death. Although animals with clinical disease are often culled, animals with subclinical paraTB cause significant economic losses because of reduced milk production and poor reproductive performance. In addition, shedding of bacteria in the faeces of these animals poses a risk for spread of the disease (Koets et al (2006), Vaccine 24; 2550-2559; Stabel (1998), J. Dairy Sci. 81; 283-288; and Jungersen et al (2002), Clin. Diagn. Lab. Immun. 9; 453-460).
Vaccination against paraTB is not widely used. Currently available vaccines are considered to have variable efficacy and although they reduce clinical disease, their capacity to limit the frequency of subclinical infection, and thus eliminate infection from populations, is very low. A major disadvantage of vaccination is that these interfere with diagnostic tests for paraTB.
Bacteriologic culture is the most definitive method of para diagnosis but is time consuming and labour intensive. There is thus a need for an improved diagnostic method for paraTB, preferably one which is able to differentiate between vaccinated and infected individuals.